The issue of whether overexpression of wild-type TDP-43 in rodent brain could be neurotoxic is neatly brought out in this paper from the group of Ronald Klein with senior authors Dennis Dickson and Mike Hutton. Using an adeno-associated virus type 9 (AAV9) vector for human TDP-43 expression by stereotactic injection into the rat substantia nigra (SN), Tatom and colleagues show that overexpression of human wild-type TDP-43 on its own can kill dopaminergic neurons in rats in a dose-dependent manner (Tatom et al., 2009).

This approach is surely welcome at a time when many laboratories are struggling to get a decent TDP-43 expression in transgenic germlines. The reason why this is problematic is made apparent by this paper, where (roughly estimated) threefold wild-type TDP-43 overexpression almost completely wipes out the targeted neurons accompanied by neurodegeneration-related astro- and microgliosis. TDP-43 was selectively expressed in neurons as AAV9 has a natural neurotropism, perhaps due to the virus capsid (Bartlett et al., 1998); and while SN is chosen for sake of...  Read more

The issue of whether overexpression of wild-type TDP-43 in rodent brain could be neurotoxic is neatly brought out in this paper from the group of Ronald Klein with senior authors Dennis Dickson and Mike Hutton. Using an adeno-associated virus type 9 (AAV9) vector for human TDP-43 expression by stereotactic injection into the rat substantia nigra (SN), Tatom and colleagues show that overexpression of human wild-type TDP-43 on its own can kill dopaminergic neurons in rats in a dose-dependent manner (Tatom et al., 2009).

This approach is surely welcome at a time when many laboratories are struggling to get a decent TDP-43 expression in transgenic germlines. The reason why this is problematic is made apparent by this paper, where (roughly estimated) threefold wild-type TDP-43 overexpression almost completely wipes out the targeted neurons accompanied by neurodegeneration-related astro- and microgliosis. TDP-43 was selectively expressed in neurons as AAV9 has a natural neurotropism, perhaps due to the virus capsid (Bartlett et al., 1998); and while SN is chosen for sake of convenience allowing a rapid estimation of neuronal loss and behavior deficit, it is not without relevance. TDP-43 is naturally expressed in this brain region, and TDP-43 pathology is observed in the nigrostriatal pathway in a variety of neurodegenerative diseases.

The descriptive neuropathology is fascinating to read. The transgenic protein predominantly homes to the neuronal nuclei, but in approximately 1 percent of the neurons, diffuse cytoplasmic TDP-43 accumulations are also observed. Occasionally, granular textures are observed that are indicative of pre-inclusion lesions as expected at 4 weeks of disease duration. One of the pathological hallmarks of diseased neurons in ALS and FTLD-TDP patients (FTLD-U in the old terminology) is redistribution of TDP-43 from its normal nuclear localization to the cytoplasm, where it is phosphorylated and ubiquitinated and forms insoluble aggregates.

Interestingly, ubiquitin labeling was also observed in neuronal cytoplasm. It is not clear what proportion of neurons were labeled or whether it co-localized with TDP-43, but given that it was not observed in the contralateral, uninjected side, it is very likely that it is the transgenic TDP-43 protein that is ubiquitinated. It will be interesting to study whether TDP-43 is phosphorylated and/or cleaved into the disease-characteristic ~25-kDa C-terminal fragments. This would further strengthen the justification of trying to develop a rodent model of TDP-43 proteinopathy.

At present the mechanism for TDP-43 dose-related neurodegeneration is unclear. Even so, considering the various important physiological functions of TDP-43 and a tight control on its expression levels, these data are not surprising. Of relevance to the majority of ALS and FTLD-TDP patients is that it is the wild-type TDP-43 protein that is accumulating and causing neurotoxicity. Given the speed at which these studies could be accomplished, it would be very interesting to study whether a similar overexpression of TDP-43 C-terminus recapitulates key features of TDP-43 proteinopathy in rat brain, as has been shown recently in cell cultures (Igaz et al., 2009).

Lastly, having an AAV-based TDP-43 rodent model is not a substitute for a rodent model with stable germline transmission achieved by either a constitutive or inducible expression system. That amongst all would allow us to understand which neurons are more vulnerable to TDP-43 gene dosage. But until these models are developed, expression of TDP-43 by AAV-mediated somatic cell transfer approaches will continue to shed light on TDP-43 mediated disease mechanisms.

References:
Bartlett JS, Samulski RJ, McCown TJ. Selective and rapid uptake of adeno-associated virus type 2 in brain. Hum Gene Ther. 1998 May 20;9(8):1181-6. Abstract

Igaz LM, Kwong LK, Chen-Plotkin A, Winton MJ, Unger TL, Xu Y, Neumann M, Trojanowski JQ, Lee VM. Expression Of TDP-43 C-terminal fragments in vitro recapitulates pathological features of TDP-43 proteinopathies. J Biol Chem. 2009 Jan 21; Abstract

Tatom JB, Wang DB, Dayton RD, Skalli O, Hutton ML, Dickson DW, Klein RL. Mimicking Aspects of Frontotemporal Lobar Degeneration and Lou Gehrig's Disease in Rats via TDP-43 Overexpression. Mol Ther. 2009 Feb 17; Abstract

View all comments by Samir Kumar-Singh

These papers represent exciting work describing a new genetic mutation associated with familial ALS. The results further highlight the importance for RNA processing in at least familial forms of motor neuron disease. Much work remains to determine the exact mechanisms by which FUS modulates motor neuron survival. It may be related to that of TDP-43. However, the lack of cytoplasmic aggregation of TDP-43, and rare ubiquitin inclusions in the patients with FUS mutations, suggest the mechanisms may be distinct. It is interesting that FUS protein did not accumulate in the cytoplasm of motor neurons in sporadic ALS patients, again suggestive that the pathogenic mechanisms of mutant FUS-induced motor neuron degeneration may be distinct from that in sporadic ALS.

View all comments by Robert Bowser

These studies raise interesting questions about whether one problem in ALS and perhaps other neurodegenerative diseases is that RNA trafficking proteins fail to properly deliver RNAs to dendritic spines. The paper by Kwiatkowski et al. reports evidence that wild-type FUS and TDP-43 may be involved in transporting RNA into dendrites, where it mediates local protein synthesis that can be stimulated by neural activity. The clumping of the mutant form described by both new papers could therefore perturb the transport of RNA. Local protein synthesis in dendrites plays a major role in the activity-dependent modulation of synaptic strength. Changes in synaptic activity have been recently reported in the mouse model of SOD1 mutation (van Zundert et al., 2008), so it will be worthwhile to examine this issue in the FUS mice that will certainly be developed by these investigators.

View all comments by Eric Frank

This is an extremely exiting story in the understanding of ALS pathogenesis. It actually it dates back to 1998—with the first description of mRNA processing errors in sporadic ALS (Lin et al., 1998), which, interestingly, was made not in the SOD1 mouse model. At the same time, the spinal muscular atrophy gene was discovered. SMA is not unlike a childhood ALS, though predominately lower motor neurons are affected in that disease. The SMA gene defect is involved in RNA metabolism. So for the next 10 years, the SMA field has investigated the pathobiology of the defective protein. At the time it made the link between sporadic ALS and the SMA story intriguing. But there was no clear genetic link (or cause for the changes in sporadic ALS).

Feed forward to 2008, when Chris Shaw and others found a true genetic defect in RNA metabolism-based protein TDP-43. (Of course more work needs to be done on that.) And now another gene by the Shaw group, and now verified by the group in Boston, does set a string of targets that all focus on RNA...  Read more

This is an extremely exiting story in the understanding of ALS pathogenesis. It actually it dates back to 1998—with the first description of mRNA processing errors in sporadic ALS (Lin et al., 1998), which, interestingly, was made not in the SOD1 mouse model. At the same time, the spinal muscular atrophy gene was discovered. SMA is not unlike a childhood ALS, though predominately lower motor neurons are affected in that disease. The SMA gene defect is involved in RNA metabolism. So for the next 10 years, the SMA field has investigated the pathobiology of the defective protein. At the time it made the link between sporadic ALS and the SMA story intriguing. But there was no clear genetic link (or cause for the changes in sporadic ALS).

Feed forward to 2008, when Chris Shaw and others found a true genetic defect in RNA metabolism-based protein TDP-43. (Of course more work needs to be done on that.) And now another gene by the Shaw group, and now verified by the group in Boston, does set a string of targets that all focus on RNA metabolism and (lower) motor neurons.

By the way, all these cases appear to predominately involve a lower motor neuron form of ALS. The hint from genetics does suggest more of a loss of function rather than gain, but cell biology will ultimately sort that out. We certainly await the generation of mouse or fly models, which are now well underway for TDP-43. However, this may be a particularly difficult target for specific, non-toxic drug therapy.

View all comments by Jeffrey D. Rothstein

These back-to-back papers on the identification of FUS (fused in sarcoma) gene as a new genetic component of ALS open a new era of research and direct our attention to mRNA biology with respect to disease. After the first identification of mRNA processing errors in ALS patients (Lin, Bristol et al., 1998), the discovery of TDP-43 (Neumann, Sampathu et al., 2006) and now the FUS gene clearly indicate the importance of mRNA management in neurodegenerative diseases. Defects in RNA transcription, splicing, and trafficking may be the reason for cell-type-specific degeneration of motor neurons in ALS. Motor neurons both in the cortex and spinal cord are very large excitatory neurons that extend long axons to their targets and require high levels of energy and protein integrity for survival and function. Defects in transcriptional mechanisms may result in splicing defects, which could give rise to formation of non-functional proteins that would deplete the pool of required proteins for cellular function, and these non-functional proteins may form aggregates that are toxic to neurons. In...  Read more

These back-to-back papers on the identification of FUS (fused in sarcoma) gene as a new genetic component of ALS open a new era of research and direct our attention to mRNA biology with respect to disease. After the first identification of mRNA processing errors in ALS patients (Lin, Bristol et al., 1998), the discovery of TDP-43 (Neumann, Sampathu et al., 2006) and now the FUS gene clearly indicate the importance of mRNA management in neurodegenerative diseases. Defects in RNA transcription, splicing, and trafficking may be the reason for cell-type-specific degeneration of motor neurons in ALS. Motor neurons both in the cortex and spinal cord are very large excitatory neurons that extend long axons to their targets and require high levels of energy and protein integrity for survival and function. Defects in transcriptional mechanisms may result in splicing defects, which could give rise to formation of non-functional proteins that would deplete the pool of required proteins for cellular function, and these non-functional proteins may form aggregates that are toxic to neurons. In addition, defects in the trafficking of mRNA may lead to depletion of key proteins that are in high demand locally for motor neuron function. But if FUS has a general function in mRNA transcription, splicing, and trafficking, why do mutations in this gene cause ALS and not other neurodegenerative diseases? What makes motor neurons more vulnerable in the presence of defective FUS? It could be true that in motor neurons FUS controls the transcription of a distinct set of mRNA that is expressed in a cell-type-specific manner in motor neurons, or that FUS controls the production of a key protein that is highly required in motor neurons when compared to other cell-types, and thus motor neurons may become vulnerable first. FUS seems to be the tip of the iceberg. Finding effectors, binding partners including mRNA, may lead to the identification of key components of both familial and sporadic ALS. More work is on the way!

References:

Kneussel M. Dynamic regulation of GABA(A) receptors at synaptic sites. Brain Res Brain Res Rev. 2002 Jun ;39(1):74-83. Abstract

Lin CL, Bristol LA, Jin L, Dykes-Hoberg M, Crawford T, Clawson L, Rothstein JD. Aberrant RNA processing in a neurodegenerative disease: the cause for absent EAAT2, a glutamate transporter, in amyotrophic lateral sclerosis. Neuron. 1998 Mar;20(3):589-602. Abstract

Neumann M, Sampathu DM, Kwong LK, Truax AC, Micsenyi MC, Chou TT, Bruce J, Schuck T, Grossman M, Clark CM, McCluskey LF, Miller BL, Masliah E, Mackenzie IR, Feldman H, Feiden W, Kretzschmar HA, Trojanowski JQ, Lee VM. Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Science. 2006 Oct 6;314(5796):130-3. Abstract

Vance C, Rogelj B, Hortobágyi T, De Vos KJ, Nishimura AL, Sreedharan J, Hu X, Smith B, Ruddy D, Wright P, Ganesalingam J, Williams KL, Tripathi V, Al-Saraj S, Al-Chalabi A, Leigh PN, Blair IP, Nicholson G, de Belleroche J, Gallo JM, Miller CC, Shaw CE. Mutations in FUS, an RNA processing protein, cause familial amyotrophic lateral sclerosis type 6. Science. 2009 Feb 27;323(5918):1208-11. Abstract

View all comments by P. Hande Ozdinler

This study elegantly gives a first insight on a transgenic mouse model of mutant TDP-43 (A315T) identified in familial ALS patients. For those in the field, it is clear that generating these mouse models is a mammoth task on its own. Among the many interesting findings in this paper, the first to catch my attention was that the 25-kDa TDP-43 C-terminal fragments (CTFs) were recovered from detergent-soluble fractions but not from urea fractions as observed in sporadic and familial ALS/FTLD patients. If the TDP-43 25-kDa CTFs would indeed be confirmed as the real culprit, this would yet again emphasize the importance of soluble but not aggregated protein/peptide in cellular toxicity, as has been shown for a number of other proteinopathies including Aβ, α-synuclein, polyglutamine expansion in Huntingtin, and mutant SOD1.

Another important observation made in this paper was that ubiquitin-immunoreactive (ir) inclusions observed in select neurons including motor neurons were not TDP-43-ir. Thus, the mutant TDP-43 (A315T) mice do not completely model ALS, where...  Read more

This study elegantly gives a first insight on a transgenic mouse model of mutant TDP-43 (A315T) identified in familial ALS patients. For those in the field, it is clear that generating these mouse models is a mammoth task on its own. Among the many interesting findings in this paper, the first to catch my attention was that the 25-kDa TDP-43 C-terminal fragments (CTFs) were recovered from detergent-soluble fractions but not from urea fractions as observed in sporadic and familial ALS/FTLD patients. If the TDP-43 25-kDa CTFs would indeed be confirmed as the real culprit, this would yet again emphasize the importance of soluble but not aggregated protein/peptide in cellular toxicity, as has been shown for a number of other proteinopathies including Aβ, α-synuclein, polyglutamine expansion in Huntingtin, and mutant SOD1.

Another important observation made in this paper was that ubiquitin-immunoreactive (ir) inclusions observed in select neurons including motor neurons were not TDP-43-ir. Thus, the mutant TDP-43 (A315T) mice do not completely model ALS, where ubiquitin-ir inclusions are also TDP-43-ir; nevertheless, this work does lead to a very interesting question: what are these inclusions composed of?

Knowing earlier studies (see Tatom et al., 2009 and ARF related news story), I am also not surprised at the glaring omission of wild-type TDP-43 mice as a better control than the non-transgenic mice utilized in this study. So although clearly not all is answered yet, let's see how these and other TDP-43 mouse models currently being developed will unfold the mysteries of TDP-43-led neurodegeneration.

View all comments by Samir Kumar-Singh